Method of tumor treatment

ABSTRACT

The present invention provides methods for treating mammalian cancer tumors, preferably solid tumors, comprising administering to a mammal in need of such treatment an effective amount of a 1,2,4-benzotriazine oxide as defined in Formula I or pharmacologically acceptable salts of said compound; and administering to the mammal from about one half hour to about twenty-four hours after administering the 1,2,4-benzotriazine oxide an effective amount of a chemotherapy agent to which the tumor is susceptible. The invention also provides kits for treatment of such tumors which comprise a chemotherapy agent and a cytotoxicity-enhancing amount of a 1,2,4-benzotriazine oxide as defined in Formula I.

This application is a continuation of application Ser. No. 08/448,705filed on May 24, 1995 now U.S. Pat. No. 5,670,502 and a continuation ofapplication Ser. No. 08/125,609 filed Sep. 22, 1993 now U.S. Pat. No.5,484,612.

FIELD OF THE INVENTION

The present invention relates to the field of treatments for cancertumors. More particularly the present invention relates to treatment ofcancer tumors with combinations of chemotherapy agents and1,2,4-benzotriazine oxides.

BACKGROUND OF THE INVENTION

The most commonly used anticancer drugs are more ctyotoxic towardnormally oxygenated tumor cells than toward hypoxic tumor cells. Hypoxiccell resistance to irradiation is also widely known. Consequently, tumorhypoxia and the resultant resistance to treatment is of concern incancer therapeutics.

Solid cancer tumors contain both adequately oxygenated cells as well asvarying proportions of inadequately oxygenated or hypoxic cells. Hypoxiausually occurs where the tumor cells are furthest away from bloodvessels. Such cells also tend to have slower rates of proliferation.Although not completely understood, resistance of hypoxic cells toanticancer drugs is generally thought to be due to inadequate uptake ofthe drug by the hypoxic cells either because they tend to be slowlygrowing or because of their distance from the blood vessels bringing thedrug. Thus, the relative proportion of hypoxic cells in the tumor can beof great importance to the outcome of the treatment. Resistant hypoxiccells that survive irradiation or drug treatment may becomereoxygenated, thereby restoring tumor sensitivity to further treatment.Nonetheless, instead of relying on uncertain events, it is desirable todevelop cancer treatments wherein cancer tumor cells, including hypoxictumor cells, are killed or rendered inactive more reliably at the timethe treatment is administered.

U.S. Pat. No. 5,175,287 issued Dec. 29, 1992 discloses the use of1,2,4-benzotriazine oxides in conjunction with radiation for treatmentof tumors. The 1,2,4-benzotriazine oxides sensitize the tumor cells toradiation and make them more amenable to this treatment modality.

Holden et al (1992) "Enhancement of Alkylating Agent Activity by SR-4233in the FSaIIC Murine Fibrosarcoma" JNCI 84: 187-193 discloses the use ofSR-4233, also known as tirapazamine, in combination with an antitumoralkylating agent. The four antitumor alkylating agents, cisplatin,cyclophosphamide, carmustine and melphalan, were each tested to examinethe ability of tirapazamine to overcome the resistance of hypoxic tumorcells to antitumor alkylating agents. Tirapazamine was tested alone andin combination with varying amounts of each of the antitumor alkylatingagents. When SR 4233 was administered just before single-dose treatmentwith cyclophosphamide, carmustine or melphalan marked dose enhancementleading to synergistic cytotoxic effects on tumor cells was observed.When SR 4233 was administered just prior to single-dose treatment withcisplatin, however, the dose enhancement lead to an additive effect,except at the highest dose level of cisplatin.

Nitroimidazole hypoxic cytotoxic agents have been combined with variousanti-cancer drugs and it was found that a therapeutic gain could beachieved when these agents were combined with various anti-cancer drugs,particularly the alkylating agents, cyclophosphamide and melphalan andthe nitrosoureas, BCNU and CCNU. However, it was later found that thetherapeutic gain produced was not the consequence of selective killingof hypoxic cells by the nitroimidazoles but appeared to be by amechanism involving the potentiation of alkylating agent-induced DNAcross-links by metabolites of the nitroimidazoles (Murray et al. (1983)Br. J. Cancer 47: 195-203)

SUMMARY OF THE INVENTION

The present invention provides methods of treating cancer tumors,particularly solid tumors comprising administering to a mammal in needof such treatment an effective amount of a compound having the formula##STR1## wherein X is H; hydrocarbyl (1-4C); hydrocarbyl (1-4C)substituted with OH, NH₂, NHR or NRR; halogen; OH; alkoxy (1-4C); NH₂ ;NHR or NRR; wherein the various R groups are independently selected fromlower alkyl (1-4C) and lower acyl (1-4C) and the R's may themselves besubstituted with OH, NH₂, alkyl (1-4C) secondary and dialkyl (1-4C)tertiary amino groups, alkoxy (1-4C) or halogen. In the case of NRR, thetwo R's can be linked together directly or through a bridge oxygen intoa morpholino ring, pyrrolidino ring or piperidino ring;

n is 0 or 1; and

Y¹ and Y² are independently either H; nitro; halogen; hydrocarbyl(1-14C) including cyclic and unsaturated hydrocarbyl, optionallysubstituted with 1 or 2 substituents selected from the group consistingof halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primaryamino (NH₂), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiaryamino, dialkyl (1-4C) tertiary amino where the two alkyls are linkedtogether to produce a morpholino, pyrrolidino or piperidino, acyloxy(1-4C), acylamido (1-4C) and thio analogs thereof, acetylaminoalkyl(1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single either (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R'O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents, orpharmacologically acceptable salts of said compound; and administeringto the mammal from about one half hour to about twenty-four hours afteradministering the compound of Formula I, as defined herein, an effectiveamount of a chemotherapy agent to which the tumor is susceptible.

The present invention also provides methods of increasing the toxicityof chemotherapy agents towards solid tumors. In this aspect of theinvention a cytotoxicity-enhancing amount of a compound of Formula I, asdefined hereinabove, is administered to a mammal having a solid tumorand in need of such treatment, the tumor further being susceptible totreatment with the chemotherapy agent, about one half hour to abouttwenty-four hours prior to administering the chemotherapy agent, orabout one hour to about two hours after aministering the chemotherapyagent.

In another aspect, the present invention provides a method of treatingmammalian cancer tumors comprising administering a compound of FormulaI, as defined herein, to the mammal one or two hours afteradministration of a chemotherapy agent.

Applicants have discovered that administering a compound of Formula I,as defined herein, either before or after the administration of achemotherapy agent surprisingly and unexpectedly killed tumor cells to amuch greater extent than administration of either agent alone, oradministration of both agents at the same time. When tirapazamine wasadministered up to twenty-four hours prior to administration ofcisplatin, Applicants found there was a ten to one thousand foldincrease in tumor cell killing above the amount of tumor cell killingfound when tirapazamine and cisplatin were administered at the sametime. The greatest synergistic effect with this combination of agentswas found when tirapazamine was administered about two and one halfhours prior to administration of cisplatin.

Applicants' claimed method represents an enormous increase in anti-tumorefficacy of the chemotherapy agent (i.e. its cytotoxic effects upontumor cells). Additionally, in tests of the systemic toxicity ofcisplatin (serum BUN and acute toxicity) the combination with theoptimum separation for tumor efficacy showed little or no enhancement ofsystemic toxicity compared to cisplatin alone. Thus, most, if not all,of the additional cell kill of the tumor cells translates into atherapeutic gain for this combination. The synergistic interactionbetween tirapazamine and cisplatin is also significant since the greatincrease in tumor cell killing was produced at a relatively low dose ofcisplatin.

The present invention is more particularly pointed out in the appendedclaims and is described in its preferred embodiments in the followingdescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of the relative clonogenic cells per tumor presentin experimental RIF-1 tumors versus time (-3 to +2 hours) ofadministration of tirapazamine relative to cisplatin.

FIG. 2 shows a graph of the relative clonogenic cells per tumor presentin experimental RIF-1 tumors versus time (-24 to 0 hours) ofadministration of tirapazamine relative to cisplatin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating mammalian cancertumors, including human cancer tumors, particularly solid tumors. Inthis aspect of the invention, an effective amount of a compound havingFormula I, as defined herein, is administered to a mammal having acancer tumor and in need of such treatment from about one half hour toabout twenty-four hours before an effective amount of a chemotherapyagent to which the tumor is susceptible is administered to the mammal.

As used herein, susceptibility of a tumor to a chemotherapy agent refersto a chemotherapty agent that is capable of exerting a therapeuticeffect on a tumor by any mechanism such as by killing tumor cells,reducing cell proliferation or reducing the size of the tumor. Also asused herein, effective amount of the compound of Formula I, as definedherein, refers to amounts capable of killing tumor cells or capable ofkilling tumor cells in conjunction with a chemotherapy agent. Aneffective amount of a chemotherapy agent refers to an amount of thechemotherapy agent capable of killing cancer cells or otherwiseproducing a therapeutic effect such as by reducing tumor size or slowingtumor cell growth and proliferation.

Another aspect of the invention provides a method for increasing thecytotoxicity of a chemotherapy agent towards a solid tumor susceptibleto treatment with the chemotherapy agent comprising, administering acytotoxicity-enhancing amount of a compound of Formula I, as definedherein, to a mammal having such a tumor from about one hour to about twohours after administering the chemotherapy agent. As used herein, theterm cytotoxicity-enhancing amount refers to an amount of the compoundof Formula I, as defined herein, that is capable of of increasing thecytotoxic effects of the chemotherapy agent on cells. Preferably thecytotoxicity-enhancing amount is sufficient to produce a synergisticeffect, i.e., greater than the sum of the effects of the chemotherapyagent and the compound of Formula I when administered singly.Cytotoxicity-enhancing amounts of the of the compound of Formula I canbe assessed by testing such compounds with a chemotherapy agent(s) in invivo and/or in vitro experimental tumor models, such as the one setforth herein, or any other tumor model known in the art. Thecytotoxicity-enhancing amount determined through in vivo and or in vitroexperimental tumor models is then used as a guide for determining theamounts of the two agents that will be administered to the mammal fortreatment of the tumor.

Another further aspect of the invention provides methods for increasingthe cytotoxicity of a chemotherapy agent towards a solid tumorsusceptible to treatment with the chemotherapy agent, comprisingadministering to a mammal having such a tumor a cytotoxicity-enhancingamount of a compound having Formula I, as defined herein, from about onehour to about two hours after administering a chemotherapy agent.

Without wishing to be bound by any theory or mode of action, at thepresent time it is believed that the combination of a benzotriazinechemotherapy agent of Formula I, as defined herein, that is specificallycytotoxic to hypoxic cancer cells and a chemotherapy agent having itsgreatest activity on normally oxygenated cancer cells provides enhancedor synergistic killing of tumor cells. The benzotriazines oxides ofFormula I, as defined herein, specifically require lower than normaloxygen concentrations in order to exert their effects. This requirementfor hypoxia is a major advantage, since it provides the basis fortumor-specific interaction between the two drugs. In general, normaltissues are at an oxygen concentration above 10-15 mm Hg. At these andhigher oxygen partial pressures, the cytotoxicity produced bytirapazamine is very low. On the other hand, many tumors have asignificant number of cells at oxygen concentrations below 10 mm Hg, atwhich partial pressures the metabolism of tirapazamine and the otherbenzotriazines of Formula I to cytotoxic species is greatly increased.As used herein hypoxic tumor cells refers to tumor cells at an oxygenpartial pressure less than about 10 mm Hg.

The methods of the present invention are useful in the treatment ofmammalian cancer tumors, including human cancer tumors, particularlysolid tumors having hypoxic regions. Examples of such tumors include,but are not limited to, adrenocarcinomas, glioblastomas (and other braintumors), breast, cervical, colorectal, endometrial, gastric, liver, lung(small cell and non-small cell), lymphomas (including non-Hodgkin's,Burkitt's, diffuse large cell, follicular and diffuse Hodgkin's),melanoma (metastatic), neuroblastoma, osteogenic sarcoma, ovarian,retinoblastoma, soft tissue sarcomas, testicular and other tumors whichrespond to chemotherapy. Thus, the methods of the present invention canbe used to treat cancer tumors, including experimentally-induced cancertumors, in any type of mammal including humans, commonly used laboratoryanimals such as rats, mice, rabbits and dogs, primates such as monkeys,and horses, cats and other animals.

The methods of the present invention can be practiced with any type ofchemotherapy agent. In any particular embodiment of the invention, thechemotherapy agent will be selected with reference to factors such asthe type of cancer tumor and the efficacy of the chemotherapy agent fortreating the cancer tumor involved. The chemotherapy agent may selectedfrom alkylating agents, antimetabolites, natural products, hormones andantagonists and other types of compounds.

Examples of alkylating agents include the nitrogen mustards (i.e., the2-chloroethylamines) such as, for example, chloromethine, chlorambucil,melphalan, uramustine, mannomustine, extramustine phosphate,mechlor-thaminoxide, cyclophosphamide, ifosamide and trifosfamide;alkylating agents having a substituted aziridine group such as, forexample, tretamine, thiotepa, triaziquone and mitomycin; alkylatingagents of the alkyl sulfonate type, such as, for example, busulfan, andpiposulfan; alkylating N-alkyl-N-nitrosourea derivatives such as, forexample, carmustine, lomustine, semustine or streptozotocine; alkylatingagents of the mitobronitole, dacarbazine and procarbazine type; andplatinum complexes such as, for example, cisplatin and carboplatin.

Examples of antimetabolites include folic acid derivatives such as, forexample, methotrexate, aminopterin and 3'-dichloromethotrexate;pyrimidine derivatives such as, for example, 5-fluorouracil,floxuridine, tegafur, cytarabine, idoxuridine, and flucytosine; purinederivatives such as, for example, mercaptopurine, thioguanine,azathioprine, tiamiprine, vidarabine, pentostatin and puromycin.

Examples of natural products include vinca alkaloids such as for examplevinblastine and vincristine; epipodophylotoxins such as, for example,etoposide, and teniposide; antibiotics such as, for example, adrimycin,daunomycin, dactinomycin, daunorubicin, doxorubicin, mithramycin,bleomycin and mitomycin; enzymes such as, for example, L-asparaginase;biological response modifiers such as, for example, alpha-interferon;camptothecin; taxol; and retinoids such as retinoic acid.

Examples of hormones and antagonists include adrenocorticoids, such as,for example, prednisone; progestins, such as, for example,hydroxyprogesterone acetate, medroxyprogesterone acetate and megestrolacetate; estrogens such as, for example, diethylstilbestrol and ethinylestradiol; antiestrogens such as for example, tamoxifen; androgens suchas, for example, testosterone propionate and fluoxymestrone;antiandrogens such as, for example, flutamide; andgonadotropin-releasing hormone analogs such as, for example, leuprolide.

Examples of miscellaneous agents include anthracenediones such as forexample, mitoxantrone; substituted ureas such as, for example,hydroxyureas; and adrenocortical suppressants such as, for example,mitotane and aminoglutethimide.

In addition, the chemotherapy agent can be an immunosuppressive drug,such as, for example, cyclosporine, azathioprine, sulfasalazine,methozsalen and thalidomide.

The chemotherapy agents useful in the practice of the present inventionare commercially available or can be prepared by methods known in theart. The chemotherapy agent can be used alone or in combination with oneor more chemotherapy agents. For example, a combination of threedifferent chemotherapy agents and one or more of the compounds ofFormula I, as defined herein, administered in accordance with themethods of the present invention could be used to treat a cancer tumor.

In the compounds of Formula I, ##STR2## X is hydrogen; unsubstitutedbranched or straight chain hydrocarbyl (1-4C) such as methyl, ethyl,s-butyl and iso-propyl; hydroxy; alkoxy (1-4C) such as methoxy, ethoxy,propoxy, and t-butoxy; primary amino (NH₂); secondary amino (NHR) whereR is an alkyl or acyl of 1 to 4 carbons, such as methylamino andethylamino; tertiary amino (NRR) where each of the R groups is an alkylor acyl of 1 to 4 carbons, for example diethylamino and the like, or thetwo R's join to form a morpholino, pyrrolidino or piperidino ring. Inthe case of the various alkyl and acyl R groups, they can be furthersubstituted with OH, NH₂, lower alkyl (1-4C) secondary amino and dialkyl(1-4C) tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy(1-4C) or halogen (fluoro, chloro, bromo or iodo) substituents.

The hydrocarbyl X groups can be further substituted with OH, NH₂, alkylsecondary amino, dialkyl tertiary amino, alkoxy (1-4C) or halogen(fluoro, chloro, bromo or iodo) substituents.

More preferably X is hydrogen, primary amino (NH₂); unsubstitutedbranched or straight chain hydrocarbyl (1-4C) or substituted branched orstraight chain hydrocarbyl (1-4C).

n is 0 or 1, preferably 1.

Y¹ and Y² are independently hydrogen; nitro; halogen (e.g. fluoro,chloro, bromo or iodo); or hydrocarbyl (1-14C). When hydrocarbyl, Y¹ andY² may be saturated or unsaturated, cyclic or acyclic, and mayoptionally be interrupted by a single ether linkage. Thus, theunsubstituted hydrocarbyl forms of Y¹ or Y² can be, for example, methyl,ethyl, n-propyl, s-butyl, n-hexyl, 2-methyl-n-pentyl, 2-ethoxyethyl,3-(n-propoxy)-n-propyl, 4-methoxybutyl, cyclohexyl, tetrahydrofurfuryl,furfuryl, cyclohexenyl, 3-(n-decyloxy)-n-propyl, and 4-methyloctyl,4,7,-dimethyloctyl.

The hydrocarbyl Y¹ and Y² groups may optionally be substituted with 1 or2 substituents selected from halogen such as fluoro, chloro, bromo oriodo; hydroxy; epoxy; alkoxy (1-4C) such as, for example, methoxy,n-propoxy and t-butoxy; alkyl thio; (1-4C) primary amino (NH₂);morpholino; pyrrolidino; piperidino; secondary amino (NHR') where R' isa 1-4C alkyl, such as methylamino, propylamino and the like; tertiaryamino (NR'R'); acyloxy and acylamino groups represented by R'COO-- andR'CONH--, respectively, and their thiol analogs represented by R'CSO--and R'CSNH-- respectively; carboxy (--C(O)OH); alkoxycarbonyl(--C--(O)OR'); carbamyl (--C(O)NH₂); alkylcarbamyl (1-4C) (--C(O)NHR');alkylsulfonyl (1-4C (R'SO₂ --); and alkyl phosphonyl (1-4C)(R'P(OR')O--).

In addition Y¹ and Y² can each independently be --NH₂, --NHR', --NR'R',--OCOR', --NH(CO)R', --O(SO)R' or --O(ROR')R' in which the various R"groups are lower alkyls (1-4C) which themselves may be substituted withOH, NH₂, alkyl secondary and tertiary amino, pyrrolidino, piperidino,alkoxy (1-4C), or halogen substituents.

More preferably, Y¹ and Y² are independently H, nitro, carboxy,alkoxycarbonyl, alkylsulfonyl or --NHR' wherein R' is --CH₂ --(CH₂)_(m)--CH₂ --NR₁ R₂, R₁ and R₂ are independently selected from the groupconsisting of hydrogen, lower alkyl, or the R₁ and R₂ groups may belinked to form a piperidino or pyrrolidino ring, and m is an integerfrom 0 to 4, preferably 1 or 2.

Particularly preferred compounds of Formula I for use in the presentinvention include 1,2,4-benzotriazine 1,4-dioxide (wherein X ishydrogen, Y¹ and Y² are each hydrogen and n is 1);3-amino-1,2,4-benzotriazine 1,4-dioxide (i.e., tirapazamine, SR 4233,wherein X is NH₂, Y¹ and Y² are each hydrogen and n is 1);3-ethyl-1,2,4-benzotriazine 1,4-dioxide (wherein X is ethyl, Y¹ and Y²are each hydrogen and n is 1); 3-propyl-1,2,4-benzotriazine 1,4-dioxide(wherein X is propyl, Y¹ and Y² are each hydrogen and n is 1) and;3-(1-hydroxyethyl)-1,2,4-benzotriazine 1,4-dioxide (wherein X is1-hydroxyethyl, Y¹ and Y² are each hydrogen and n is 1); mostparticularly 3-amino-1,2,4-benzotriazine 1,4-dioxide.

Pharmaceutically acceptable salts of the compounds of Formula I, asdefined herein, include salts formed from inorganic acids such ashydrochloric, hydrobromic, or phosphoric acids, organic acids such asacetic acid, pyruvic acid, succinic acid, mandelic acid, and p-toluenesulfonic acid; salts formed from inorganic bases such as sodium,potassium or calcium hydroxide or from organic bases such as caffeine,ethylamine or lysine.

The compounds of Formula I, as defined herein, may be administered topatients orally or parenterally (intravenously, subcutaneously,intramuscularly, intraspinally, intraperitoneally, and the like). Whenadministered parenterally the compounds will normally be formulated in aunit dosage injectable form (solution, suspension, emulsion) with apharmaceutically acceptable vehicle. Such vehicles are typicallynontoxic and nontherapeutic. Examples of such vehicles are water,aqueous vehicles such as saline, Ringer's solution, dextrose solution,and Hank's solution and nonaqueous vehicles such as fixed oils (e.g.,corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropylmyristate. Sterile saline is a preferred vehicle. The vehicle maycontain minor amounts of additives such as substances that enhancesolubility, isotonicity, and chemical stability, e.g., antioxidants,buffers, and preservatives. When administered orally (or rectally), thecompounds will usually be formulated into a unit dosage form such as atablet, capsule, suppository or cachet. Such formulations typicallyinclude a solid, semisolid or liquid carrier or diluent. Exemplarydiluents and vehicles are lactose, dextrose, sucrose, sorbitol,mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoabutter, oil of theobroma, alginates, tragacanth, gelatin,methylcellulose, polyoxyethylene, sorbitan monolaurate, methylhydroxybenzoate, propyl hydroxybenzoate, talc and magnesium stearate.

The chemotherapy agent is administered to the mammal by conventionalroutes appropriate for the particular chemotherapy agent. Thechemotherapy agent and the compound of Formula I, as defined herein, canbe administered by the same route, or by different routes, depending onthe particular combination of compound of Formula I, as defined herein,and chemotherapy agent. The compound of Formula I, as defined herein,can be administered to the mammal alone or in combination with one ormore other compounds of formula I, as defined herein.

The compounds of Formula I, as defined herein, are administered to themammal in amounts effective to kill or produce cytotoxic effects uponhypoxic tumor cells. The amount of the compound administered will dependon such factors as the type of cancer tumor, the age and health of themammal, the maximum tolerated and/or lethal dosage of the chemotherapyagent and the compound of Formula I, and the interaction of the compoundof Formula I with the chemotherapy agent. In a presently preferredembodiment of the invention, tirapazamine is administered in amounts offrom about 10 mg/m² to about 450 mg/m² ; more preferably from about 20mg/m² to about 350 mg/m² ; most preferably from about 30 mg/m² to about250 mg/m². When the compound of Formula I is administered to the mammalin divided doses, the lower dosage range may be preferably, depending onthe maximum tolerated dosage of the compound and the interaction of thecompound with the chemotherapy agent.

The chemotherapy agent is administered to the mammal in amountseffective to treat susceptible tumors. Such amounts are well-known inthe art and can be ascertained by reference to product literaturefurnished by the supplier of the chemotherapy agent or scientificliterature. In preferred embodiments of the invention, the chemotherapyagent and the compound of Formula I have a synergistic interaction uponthe tumor and it may be possible to administer the chemotherapy agent atdoses that are lower than doses recognized as effective when thechemotherapy agent is administered alone. Such lower dosages may bedesirable if the chemotherapy agent produces severe side effects in themammal to which it is administered. If the chemotherapy agent is to beadministered to the mammal in divided doses, sufficient amounts of thecompound of Formula I, as defined herein, is administered to the mammalso that the synergistic effect of the combination of two agents ismaintained, whether before the initial dose of the chemotherapy agent orprior to each individual dose of the chemotherapy agent. The methods ofthe invention can also be employed in conjunction with other types ofcancer treatments such as radiation therapy and surgical removal of thetumor.

The compound of Formula I is administered to the mammal from about onehalf hour to about twenty-four hours prior to administration of thechemotherapy agent. Alternatively, the compound of Formula I can beadministered to the mammal from about one to about two hours after theadministration of the chemotherapy agent. For some combinations ofchemotherapy agent and compound of formula I it may be possible toadminister the compound of Formula I more than twenty-four hours priorto administration of the chemotherapy agent and still retain theadvantages of the methods of the present invention. The timedifferential providing the most advantageous increase in cell toxicitycan be determined by testing the combination of compound of formula Iand chemotherapy agent in in vivo and or in vitro experimental tumormodels, such as the one set forth herein, or any other tumor model. Thetime differential determined in such models is then used as a guide fortreatment of tumors in mammals, with adjustments made during treatmentif necessary. Applicants have found that for the combination oftirapazamine and cisplatin, the greatest interaction between the twoagents was observed when tirapazamine was administered between about oneand three hours prior to administration of the cisplatin, with thegreatest increase in cell death occurring when tirapazamine wasadministered about two and one half hours prior to cisplatin. Whentirapazamine was administered one to two hours after administration ofcisplatin, an enhanced cytotoxic effect was observed, however, theincrease was not as large. In some embodiments of the invention, it maybe desirable to administer the compound of Formula I at the same time asthe chemotherapy agent.

The present invention also provides kits for treatment of mammaliantumors comprising at least one chemotherapy agent and at least onecompound of formula I, as defined herein. The compound of Formula I asdefined herein is preferably supplied in the kits incytotoxicity-enhancing amounts or doses. Suitable dosage forms for thecompounds of Formula I, as defined herein, as disclosed herein. Theparticular dosage form of the chemotherapy agent and the compound ofFormula I, as defined herein, will be determined by the type of cancertumor to be treated, the preferred route of administration and the typeof chemotherapy agent. The chemotherapy agent and the compound ofFormula I, as defined herein, are preferably supplied in separatecontainers to facilitate administration of the chemotherapy agent andthe compound of Formula I at different times in accordance with themethods of the invention.

The compounds of Formula I useful in the practice of the presentinvention can be prepared according to the methods disclosed in U.S.Pat. No. 5,175,287 issued Dec. 29, 1992, the disclosures of which arehereby incorporated by reference. General methods for preparing some3-amino derivatives can be found, for example, in Ley et al., U.S. PatNo. 3,980,779.

The compounds are prepared from benzofuroxan of the Formula: ##STR3## byreaction with a salt of cyanamide, followed by acidification of thereaction mixture. The benzofuroxan starting material is not symmetricwith respect to its own 5 and 6 positions (which are the 6 and 7positions of the resulting 3-amino benzotriazine oxide). Therefore, amixture of the 6- and 7-substituted materials may result. If desired,this mixture can be separated using conventional means into individualcomponents having a substituent in either the 6 or 7 position.

The dioxide may also be prepared from parent monoxide or1,2,4-benzotriazine by peracid oxidation (see Robbins et al, J Chem Soc3186 (1957) and Mason et al, J Chem Soc B 911 (1970)).

In addition, the monoxide may be prepared by:

(1) cyclization of a 1-nitro-2-aminobenzene compound using H₂ NCN.2HCl;

(2) oxidation of the parent compound given by the structure ##STR4## orby controlled reduction of the corresponding dioxide (see Mason, supra,and Wolf et al, J Am Chem Soc 76:355(1954)).

The 1,2,4-benzotriazines may be prepared by cyclization of formazanprecursors using BF₃ /AcOH (see Scheme I and Atallah and Nazer,Tetrahedron 38:1793 (1982)).

The 3-Amino-1,2,4-benzotriazines may be prepared either by cyclizationof a parent compound (see Scheme II and Arndt, Chem. Ber. 3522 (1913))or by reduction of the monoxide or dioxide as above.

The 3-hydroxy-1,2,4-benzotriazine oxides may be prepared using peroxideand sodium tungstate (Scheme III), a novel synthetic procedure formaking the 3-hydroxy-1,4-dioxide compound, or concentrated sulfuric acidand sodium nitrate (Scheme IV). ##STR5##

1,2,4-Benzotriazine oxides unsubstituted at the 3 position (sometimesreferred to herein as the "3-desamino" compounds) can be prepared by thefollowing method. The method involves treating a 1,2,4-benzotriazineoxide of Formula(I), wherein X is NH₂, with a lower alkyl nitrite underreductive deaminating conditions. By "reductive deaminating conditions"is meant reaction conditions which will give rise to at least about 10%,preferably at least about 50%, of the desired 3-unsubstituted reactionproduct. A preferred lower alkyl nitrite for use in said method ist-butyl nitrite. Exemplary reductive deaminating conditions involvereaction in a compatible solvent, e.g., dimethyl formamide, at atemperature of at least about 60° C., typically at a temperature in therange of 60°-65° C. This reaction is illustrated generally at Scheme V.,##STR6##

EXAMPLES

The methods of the present invention are exemplified by the followingnon-limiting examples. Examples 1-18 relate to synthesis of compounds ofFormula I, as defined herein. Example 19 relates to in vitro and in vivotests of tirapazamine and cisplatin.

EXAMPLE 1 Preparation of 3-Hydroxy-1,2,4-Benzotriazine 1,4-Dioxide##STR7##

A stirred mixture of 1.50 g (9.25 mmole) of 3-amino-1,2,4-benzotriazine1-oxide (1), 100.0 ml acid, and 30.0 ml of 30% hydrogen peroxide wastreated with 3.05 g (9.25 mmole) of Na₂ WO₄.2H₂ O. The mixture wasstirred in an oil bath at 60° C. for 4 days. The yellowish orangemixture was cooled to about 30° and filtered to remove a light yellownon-UV absorbing solid. The orange solution of hydrogen peroxide inacetic acid was evaporated to semi-dryness carefully with severaladditions of water and acetic acid to remove most of the peroxide. Theconcentrated solution was allowed to stand at room temperature to affordfour crops of an orange solid, 0.87 g (42% yield of the sodium salt of2).UV:λ_(max) (20% CH₃ OH/H₂ O): 262.2 (ε39,460); 477 (ε7,030). IR(neat): 3530 m, 3150 m, 2650 m, 2180 m and 1635 m. Anal. (calculated forthe sodium salt): C₇ H₄ N₃ O₃ Na 1.25H₂ O, 223.64; C,37.6; H,2.93;N,18.79. Found: C,37.8; H,2.75; N,18.65. ##STR8##

EXAMPLE 2 Preparation of 3-Amino-7-Trifluoromethyl-1,2,4-Benzotriazine1-Oxide ##STR9##

A mixture of 4-chloro-3-nitrobenzotrifluoride (Aldrich, 2.70 g, 12.9mmole) and cyanamide dihydrochloride (2.75 g, 24 mmole) (previouslyprepared by treating an ether solution of cyanamide with HCl gas andcollecting the precipitated solid) was heated at 140° C. for 1 hour. Theresidue was treated with 2N NaOH (45 ml), heated for a further 5 min.,and then allowed to cool. The precipitate was collected, washed with H₂O, dried, and triturated with acetone-toluene to yield 1.32 g (45%) of 3as a light yellow solid M.P. 301°-302°. TLC R_(f) 0.60 (9:1 methylenechloride: methanol on silica gel plates). MS: m/z (relative intensity)230 (100, M⁺).

EXAMPLE 3 Preparation of 3-Amino-7-Decyl-1,2,4-Benzotriazine ##STR10##

Preparation of 4-(1-decyl)-2-nitroaniline: Acetic anhydride (400 ml) wasadded over a 30-minute period to a stirred solution of 4-decylaniline(Aldrich, 80 g, 0.34 mole) in hexanes (2.41). After stirring for 1 h,the mixture was cooled and treated over 30 min. at 5°-10° C. with 70%nitric acid (34 ml). Stirring was continued at 5°-10° C. for 1 h and at25° C. for 16 h. The mixture was diluted with H₂ O(11), stirred for 5 h,poured into an open dish and allowed to stand for 16 h. After furtherdilution with H₂ O(1.51), the solid was collected and recrystallizedfrom an 85% ethanol solution (in water) to give 92 g (84%) of theintermediate as an orange solid, m.p. 64° C.

A solution (100 ml) of 85% KOH (19 g, 0.288 mole) in H₂ O was combinedwith a suspension of 4-(1-decyl)-2-nitroaniline (89 g, 0.28 mole),prepared above, in methanol (900 ml). The mixture was stirred for 6 h,neutralized to pH 7-8 with concentrated HCl, and evaporated in vacuo tonear dryness. After dilution with H₂ O (400 ml), the solid was collectedand air-dried to give 77 g (100%) of the intermediate as an orangesolid, m.p. 59° C.

1.0 g (8.7 mmole) of cyanamide dihydrochloride (previously prepared foruse by treating an ether solution of cyanamide with HCl gas andcollecting the precipitated solid) was added portionwise over 10 min toa preheated melt (190° C.) of 4-(1-decyl)-2-nitroaniline prepared in thepreceding step (500 mg, 1.8 mmole). The reaction mixture was heated at190° C. for 5 min, cooled to 25° C., treated with 6N KOH (10 ml), andheated at 90°-95° C. for 1 h. After cooling to 25° C., the solid wascollected, washed with H₂ O and ethanol and air dried to give 0.25 g(46%) of compound 4 as a light yellow solid, m.p. 177° C. (dec). MS: m/z(relative intensity) 285 (100, M⁺), 302 (13)

EXAMPLE 4 Preparation of 3-Amino-7-Carbamyl-1,2,4-Benzotriazine 1-Oxide##STR11##

Preparation of 4-chloro-3-nitrobenzamide: 20.2 g (0.1 mole) of4-chloro-3-nitrobenzoic acid (Aldrich) and thionyl chloride (20 ml) werecombined, allowed to stand for 16 h, and refluxed for 4 h to give aclear red solution. The solution was evaporated in vacuo and azeotropedwith benzene. The residue was dissolved in acetonitrile (20 ml) andadded over 30 min to cold (-10° C.) concentrated ammonium hydroxide (100ml). After 3 h at -10° C. and 16 h at 25° C. the mixture was poured intoan open dish and allowed to evaporate to dryness. The residue wasslurried in H₂ O and the solid was collected and air-dried to give 19.8g (98%) of the intermediate as a light yellow solid, m.p. 153° C.

A solution of Na (3.45 g, 0.15 mole) in ethanol (75 ml) was added to asolution of guanidine hydrochloride (15.8 g, 0.165 mole) in ethanol (75ml). After 1 h the mixture was filtered and the filtrate was combinedwith a suspension of 4-chloro-3-nitrobenzamide (10 g, 0.05 mole)prepared above, in ethanol (50 ml). The mixture was stirred and refluxedfor 16 h, cooled to 0°-5° C., and acidified with concentrated HCl (8ml). The collected solid was combined with K₂ CO₃ (28 g, 0.2 mole) andH₂ O (40 ml) and the mixture was stirred and heated at 100° C. for 8 h.After cooling to 25° C., the solid was collected, washed with H₂ O, andair-dried. The solid was suspended in boiling ethyl acetate, collectedand washed with hot ethyl acetate. The solid was repeatedly suspended inboiling dioxane and collected (6×100 ml). The combined filtrate wasevaporated in vacuo to a solid. The solid was suspended in 95% ethanol,collected and air-dried to give 0.44 g (4.3%) of compound 5 as a lightyellow solid, m.p. 300° C. TLC: Rf=0.23 (methylene chloride: acetone of2:1, silica gel plates). MS: m/z (relative intensity) 205 (100, M⁺).

EXAMPLE 5 Preparation of 7-Acetyl-3-Amino-1,2,4-Benotriazine 1-OxideOxime ##STR12##

A combined mixture of 7-acetyl-3-amino-1,2,4-benzotriazine 1-oxide(prepared in Example 5; 50 mg, 0.25 mmole), hydroxylamine hydrochloride(200 mg, 2.88 mmole), pyridine (1 ml), and ethanol (1 ml) was heated at90°-95° C. for 1 h and then cooled to 25° C. The mixture was dilutedwith 95% ethanol (5 ml) and the solid was collected and air-dried togive 30 mg (56%) of compound 6 as a light yellow solid, m.p. 278° C.(dec.). TLC: R_(f) =0.60 (9:1 methylene chloride: methanol). MS: m/z(relative intensity) 219 (100, M⁺).

EXAMPLE 6 Preparation of 3-Amino-6(7)-Decyl-1,2,4-Benzotriazine1,4-Dioxide ##STR13##

5-(1-decyl)-benzofuroxan: A combined mixture of4-(1-decyl)-2-nitroaniline (77 g, 0.28 mole), 5.25% NaOCl in H₂ O (476g, 0.34 mole), 85% KOH (20.3 g, 0.31 mole), nBu₄ NHSO₄ (4.7 g, 0.014mole), and CH₂ Cl₂ (2.28 l) was stirred rapidly for 6 h and diluted withH₂ O (500 ml) and CH₂ Cl₂ (1 l). The separated organic phase was washedsuccessively with 1N HCl (1 l) and brine (2×1 l)), dried (Na₂ SO₄), andconcentrated in vacuo to yield a red oil, 70 g (92%).

A solution of 5-(1-decyl)-benzofuroxan as prepared above (10 g, 0.036mole) and benzyltriethyl ammonium chloride (0.36 g, 0.0016 mole) in DMSO(180 ml) was treated gradually over several hours with cyanamide (13.0g, 0.31 mole) and K₂ CO₃ (36.8 g, 0.27 mole). The mixture was stirredfor 48 h and filtered. The filtrate was diluted with H₂ O (6 l) andglacial acetic acid (40 ml) and extracted with CH₂ Cl₂ (4×500 ml). Thecombined organic solution was washed successively with 5% NaHCO₃solution (1×500 ml) and brine (2×500 ml), dried (Na₂ SO₄), andevaporated in vacuo to dryness. The crude product was purified bychromatography on silica gel using CH₂ Cl₂ : methanol (98.2) to give 1.8g (16%) of compound 7 as a red solid, m.p. 155° C. (dec). MS: m/z(relative intensity) 318 (4, M⁺), 285 (100).

EXAMPLE 7 Preparation of 1,2,4-Benzotriazine Dioxide ##STR14##

A mixture of 1.80 g (13.73 mmole of 90% H₂ O₂ (9 ml), trifluoroaceticanhydride (13.5 ml) and Na₂ WO₄.2-H₂ O (12.50 g, 38 mmole) in CHCl₃ (170ml) was stirred at room temperature for 5 days. The reaction mixture wasdiluted with H₂ O (100 ml) and extracted with CHCL₃ (100 ml). Theorganic layer was washed with H₂ O (50 ml), dried (Na₂ SO₄), and thesolvent removed in vacuo. The residue was chromatographed on silica gelusing EtOAcCH₂ Cl₂ (1:1) to give 0.30 g (13.4%) of compound 9 as ayellow solid, m.p. 204°-205° C. Anal. Calc'd. for C₇ H₅ N₃ O₂ (163.13):C, 51.5: H, 3.09; N, 25.76. Found: C, 51.6; H, 3.36; N, 26.01. MS: m/z(relative intensity) 163 (100, M⁺), 147 (50). TLC: Rf=0.27 (EtOAc--CH₂Cl₂, 1:1, silica gel plates). IR (nujol): 1600 μ, 1460 μ, 1300 μ, UV:λ_(max) (H₂ O); 227 (e22,900) 252 (e12,950): 392 (e4,080).

EXAMPLE 8 Preparation of 7-Chloro-3-Hydroxy-1,2,4-Benzotriazine1,4-Dioxide ##STR15##

A mixture of 1.50 g (7.63 mmole) of 10 in 100 ml acetic acid was treatedwith 2.52 g (7.63 mmole) of Na₂ WO4.2H₂ O and 30 ml of 30% H₂ O₂. Themixture was stirred and heated for 6 days at 50° C., then slowlyevaporated to dryness to remove H₂ O₂. The residue was boiled in 250 mlH₂ O and filtered to remove about 25 mg of starting material 10. Theaqueous solutions were extracted with 2×250 ml portions of ethylacetate. A deep red crystalline material that was characterized as 12 byTLC and Mass. Spec. analysis formed in the partitioning mixture aboveand was collected by filtration to afford 60.0 mg of a yellowish orangesolid (3.7% yield), characterized as follows as 12, which showed goodsolubility in a mixture of hot isopropyl alcohol and water. Mass. Spec.:M⁺ =212 (q=100) (compound 10); TLC: R_(f) =0.34 (acetone, silica gelplates).

The ethyl acetate solutions above, separated from the H₂ O layer afterthe filtration to remove 12, were evaporated to dryness. The residue wasthen treated with isopropyl alcohol at room temperature to afford a dullorange solid, 0.41 g (25% yield) of 11. Mass. Spec.: M⁺ =213 (q=70);TLC: R_(f) =0.22 (acetone, silica gel plates). Compound 11 wascharacterized as the ammonium salt, C₇ H₄ ClN₃ O₃ NH₃, m.w. 230.61, asfollows. The free acid 11 was dissolved in concentrated NH₄ OH and thenchilled in ice and filtrated to remove a trace of insoluble 12. The redfiltrate and washings were evaporated to dryness, leaving areddish-orange solid. The solid was treated with 50 ml of boiling1,2-dimethoxyethane, collected on a filter and washed with an additional25 ml of hot 1,2-dimethyl ether. The solid was dried over P₂ O₅ at 56°C.1/1.0 mm, leaving 0.244 g (87% yield) of 13 ##STR16## Anal. Calc'd.for C₇ H₄ ClN₃ O₃ NH₃ (230.61): C, 36.5; H, 306; N 24.30 Found: C 36.5;H3.07; N 23.94 UV: λ_(max) (H₂₀): 219 (ε12,580); 265.4 (ε40,000);4830486 (ε6.640).

EXAMPLE 9 Preparation of 7-Nitro-3-Amino-1,2,4-Benzotriazine 1,4-Dioxide##STR17##

7-Nitro-3-trifluoroacetamido-1,2,4-benzotriazine 1-oxide (15): Asolution of 7-nitro-3-amino-1,2,4-benzotriazine 1-oxide (14) (4.00 g,19.3 mmol; Parish Chemical Co.), CHCl₃ (125 ml) and trifluoroaceticanhydride (12.0 ml, 85.0 mmol) was stirred at room temperature for 44hr. The resultant light yellow solid was filtered, washed with CHCl₃ (50ml) and dried to give 5.35 g (91% (yield) of the product as a yellowsolid. Anal. Calc'd for C₉ H₄ F₃ N₅ O₄ : C, 35.7; H, 1.33; N, 23.10.Found: C, 35.7; H, 1.23; N, 23.06.

7-Nitro-3-amino-1,2,4-benzotriazine 1,4-oxide (16): To a stirredsolution of 7-nitro-3-trifluoroacetamide-1,2,4-benzotriazine 1-oxideprepared above (15) (2.50 g, 8.25 mmol) in CHCl₃ (200 ml) was added Na₂WO₄.2 H₂ O (90 mg, 0.273 mmol) followed by 70% H₂ O₂ (10 ml). After 15min the solution was treated with trifluoroacetic anhydride (8.0 ml,56.7 mmol) and stirring was continued at room temperature for 64 hr. Thereaction mixture was chromatographed (EtOAc, 20% MeOH/acetone, andfinally 20% DMF/acetone) then recrystallized in acetone to give 1.20 g(65% yield) of the product (16) as an orange solid, mp 286°-288° C.(dec.). UV: λ_(max) 259, 300, 345, 387, 472. Anal. Calc'd. for C₇ H₅ N₅O₄ C, 37.70; H, 2.26; N, 31.39. Found: C, 7.70; H, 2.13; N, 30.94.

EXAMPLE 10 Preparation of3-(3-N,N-Diethylaminopropylamino)-1,2,4-Benzotriazine 1,4-Dioxide##STR18##

3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1-oxide (18): Asolution of 3-chloro-1,2,4-benzotriazine 1-oxide (17) (3.0 g, 16.5 mmol)(produced by the method of Sasse et al., U.S. Pat. No. 4,289,771) in CH₂Cl₂ (100 ml) was treated with N,N-diethyl-propylenediamine (9.5 ml, 88.3mmol). After 20 hr at room temperature the mixture was diluted with1,2-dichloroethane (50 ml) and washed successively with Na₂ CO₃ and H₂O. The yellow solution was dried (Na₂ SO₄), filtered and evaporated invacuo to give 3.93 g (87% yield) of the product as a yellow Solid.Recrystallization (ether/petroleum ether) yielded pure material, m.p.47°-48° C. Anal. Calc'd. for C₁₄ H₂₁ N₅ O (18): C, 61.10; H, 7.69; N,25.44. Found: C, 61.30; H, 7.80; N, 25.61.

3-3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1,4-oxide (18a): Toa stirred solution of3-(3-N,N-diethylaminopropylamino)-1,2,4-benzotriazine 1-oxide 18prepared as above ((1.60 g, 6.10 mmol) in CHCl₃ (50 ml) was addedtrifluoroacetic anhdride (22.0 ml). After 15 min the mixture was cooledto -10° C., 70% H₂ O₂ (10 ml) added and then stirred at room temperaturefor 20 days. The reaction mixture was dried (Na₂ SO₄), filtered andevaporated to dryness. The residue was dissolved in saturated NaHCO₃solution (50 ml) and extracted with CH₂ Cl₂ (3×150 ml). The organiclayer was dried (Na₂ SO₄), filtered and evaporated to give the product18a, 0.51 g (29% yield) as a red solid. m.p. 92°-94° C. NMR: δ (400 MHz,CDCl₃) 1.11 (6H, t, J=7.1 Hz, CH₃), 1.84-1.90 (2H, m, H-2'), 2.48-2.64(4H, m, NCH₂ CH₃, and H-3'), 3.68 (2H, br t, J=5.5 Hz, H-1'), 7.46 (1H,ddd, J=7.1, 7.0 and 1.2 Hz, H-6), 7.84, ddd, J=7.0, 6.9 and 1.2 Hz,H-7), 8.31 (2H, m; H-5 and 8), 8.80 (1H, br s, NH), UV: λ_(max) 220,270, 476. Anal. Calc'd for C₁₄ H₂₁ N₅ O₂. (1/3 H₂ O): C, 56.50; H, 7.34;N, 23.55. Found: C, 56.90; H, 7.15; N, 23.40.

EXAMPLE 11 Preparation of7-Nitro-3-(2-N,N-Diethylaminoethylamino)-1,2,4-Benzotriazine 1,4 Dioxide##STR19## 7-Nitro-3-(2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine1-oxide hydrochloride (20): A solution of7-nitro-3-chloro-1,2,4-benzotriazine 1-oxide 919) (1.60 g, 7.06 mmol)(prepared as generally shown in Sasse et al, U.S. Pat. No. 4,289,771,with (a) NaNO₂ and H₂ SO₄ at 40° C., followed by (b) chlorination withPOCl₃ at 106° C.) in CH₂ Cl₂ (50 ml) was treated withN,N-diethylethylenediamine (6.0 ml, 42.7 mmol). After 6 hr at roomtemperature the mixture was evaporated to dryness under high vacuum at60° C. The yellow solid was stirred in 20% iPrOH/ether (150 ml) for 5hr, filtered, washed with iPrOH then petroleum ether and dried (80°C./1.0 mmHg) to give 1.80 g (74% yield) of the product 20 as yellowneedle crystals. NMR δ (90 MHz, d₆ -DMSO/d₄ -MeOH) 1.25 (6H, t, J=6.0Hz, CH₃), 3.25 (6H, m, NCH₂), 3.82 (2H, m, H-1'), 7.74 (1H, d, J=7.0 Hz,H-5), 8.52 (1H, dd, J=7.0 and 2.0 Hz, H-6), 8.91 (1H, d, J=2.0 Hz, H-8).

7-Nitro-3-2-N,N-diethylaminoethylamino)-1,2,4-benzotriazine 1,4-dioxidehydrochloride (21): To a stirred solution of7-nitro-3-(2-N,N-diethylaminoethylamino) 1,2,4-benzotriazine 1-oxide(20; prepared as described above) (0.50 g, 1.46 mmol) in CHCl₃ (50 ml)at 0° C. was added trifluoroacetic anhydride (9.0 ml). After 30 min 70%H₂ O₂ (4.0 ml) was added and the mixture stirred at room temperature for3 days, then dried (Na₂ SO₄), filtered, and evaporated in vacuo todryness to give the trifluoroacetate salt 0.67 g (45% yield). Thisproduct was dissolved in saturated NaH--CO₃ solution (30 ml) andextracted with CH₂ Cl₂ (3×30 ml). The dichloromethane was washed with H₂O, dried (Na₂ SO₄), filtered, saturated with gaseous HCl and evaporatedto dryness to give 0.35 g (63% yield, 28% overall) of the product as ared solid, m.p. 194°-195° C. UV: λ_(max) 260, 306, 388, 479. Anal.Calc'd. for C₁₃ H₁₈ N₆ O₄ HCl: C, 43.50; H, 5.34; N, 23.43. Found: C,43.20; H, 5.37; N, 23.11.

The following Examples 12-15 are directed to reductive deaminationreactions for preparing compounds of Formula (I) which are unsubstitutedat the 3-position, i.e., wherein the substituent "X" is hydrogen.

EXAMPLE 12 Preparation of 1,2,4-Benzotriazine 1,4-Dioxide by ReductiveDeamination of 3-Amino-1,2,4-Benzotriazine 1,4-Dioxide ##STR20##

To a rapidly stirred solution of t-butyl nitrite (867 mg, 1.0 ml, 8.41mmol) in DMF (20 ml) at 60°-65° C. was added 3-amino-1,2,4-benzotriazine1,4-dioxide ("SR 4233") (500 mg, 2.81 mmol) (prepared by the method ofSeng et al., Anqew. Chem. Internat. Edit. 11 (1972)) in small portionsover 5 min. Following the addition, and subsidence of the concomitanteffervescence (approx. 5 min), the solution was collected and reducedunder high vacuum to a dark waxy solid. Flash chromatography (30%EtOAc/CH₂ Cl₂) gave a yellow solid, m.p. 188°-189.5° C. (dec.), whichwas recrystallized from ethanol to give 195 mg (43% yield) of theproduct 9 as bright yellow platelets, m.p. 192°-194° C. (dec.). NMR:(400 MHz, d6-acetone) 8.04 (1 H, ddd, J=8.5, 7, 1.5 Hz), 8.15 (1 H, ddd,J=8.5, 7, 1.5 Hz), 8.42 (1 H, dd, J--8.5, 1.5 Hz), 8.43 (1 H, dd, J=8.5,1.5 Hz) 9.05 (1 H, s, H-3). UV: λ_(max) 405, 300, 225. MS: m/z (relativeintensity), 147(13), 136(19), intensity) 164(9), 163(100, M+), 147(13),136(19), 90(7), 78(27), 76(26), 75(8) 64(9), 63(10), 52(12), 51(48),50(28), 38(8), 37(5), 30(18), 28(6), 27(7). Anal. Calc'd. for C₇ H₅ N₃O2: C, 51.54; H, 3.09; N, 25.76. Found C, 51.42; H, 3.02; N, 25.66.

EXAMPLE 13 Preparation of 7-Allyloxy-1,2,4-Benzotriazine 1,4-Dioxide ViaReductive Deamination ##STR21##

7-Allyloxy-1,2,4-benzotriazine 1,4-dioxide (24): To a stirred solutionof t-butyl nitride (271 mg, 0.312 ml, 2.63 mmol) in DMF (15 ml) at60°-65° C. was added 7-allyloxy-3-amino-1,2,4-benzotriazine 1,4-dioxide23 (205 mg, 0.875 mmol) in small portions over 5 min. After 30 minadditional t-butyl nitrite (271 mg, 0.312 ml, 2.63 mmol) was added, andshortly thereafter the deep red solution effervesced and lightenedappreciably in color over a period of a few minutes. After an additional30 min the resultant orange solution was reduced under vacuum to a brownsolid which was sequentially flash chromatographed (10% EtOAc/CH₂ Cl₂)and crystallized (CH₂ Cl₂ /petroleum ether) to give 72 mg (38% yield) ofthe product 24 as light orange crystals, m.p. 147°-148° C. NMR: δ(400MHz, d₆ -acetone) 4.89 (2H, ddd, H-1', J_(1'2') =5.5, J_(1').3'cis=J_(1').3'trans =1.5 Hz), 5.36(1 H, ddd, H-3', J_(3').2'cis =10.5,J_(3').3' =1.5 Hz), 5.52 (1 H, ddd, H-3', J_(3').2'trans =17.5,J_(3').3' =3, J_(3'1') =1.5 Hz), 6.14 (1 H, ddd, H-2', J_(2'),3'cis=10.5, J_(2').1' --5.5 Hz), 7.70 (1 H, d, H-8, J₈.6 =2.5 Hz), 7.74 (1 H,dd, H-6, J₆.5 =9.5, J₆.8 --2.5 Hz), 8.33 (1 H, d, H-5, J₅.6 =9.5 Hz),8.93 (1 H,s, H-3). UV: λ_(max) 425, 410, 365, 355, 320, 245, 200. MS m/z(relative intensity) 220(4), 219(34 M+), 103(4), 77(4), 75(4), 63(13),62(4), 42(3), 41(100), 39(16). Anal. Calc'd. for C₁₀ H₉ N₃ O₃ : C,54.79; H, 4.14; N, 19.17. Found: C, 54.73; H, 4.16; N, 19.15.

EXAMPLE 14 Preparation of7-(3-N-Ethylacetamido-2-acetoxypropoxy)-1,2,4-Benzotriazine 1,4-DioxideReductive Amination ##STR22##

To a stirred solution of t-butyl nitrite 9185 mg, 1.79 mmol) in DMF (5ml) at 60° C. was added via syringe a solution of7-(3-N-ethylacetamido-2-acetoxypropoxy)-3-amino-1,2,4-benzotriazine1,4-dioxide (25) (125 mg, 0.329 mmol) in DMF (5 ml) over a period of 1min. After 5 min additional t-butyl nitrite (217 mg, 2.10 mmol) wasadded and an intermediate reaction occurred, as evidenced by theevolution of a gas and a change in color of the solution from red tolight orange. After an additional 10 min the solution was stripped to ayellow/brown solid and eluted through silica gel with 5% MeOH/CH₂ Cl₂ togive CH₂ Cl₂ /ligroin gave 90 mg yellow solid (75% yield), m.p.179°-180.5° C. NMR: δ(400 MHZ, d₄ -methanol, mixture of rotamers, ratioapprox. 2:1) 1.12, 1.22 (t's, 1:2,3 H total, J=7 Hz), 2.0-6, 2.07 (s's,2:1, 3 H total), 2.11, 4.34-4.48 (m, 2 H), 5.48-5.58(m, 1 H), 7.76-7.86(m, 2H), 8.36-8.42 (m, 1 H), 9.04, 9.06 (s's, 2:1, 1 H total). UV:λ_(max) 420, 405, 365, 350, 315, 240, 200. MS: m/z (relative intensity)365(0.5), 364(1.4, M+), 349(0.5), 348(1.1), 347(0.5), 332(1.2),331(3.6), 187(7), 186(66), 102(6), 100(21), 84(30), 63(6), 58(100),56(8), 43(65), 42(9), 41(9), 41(5), 30(14), 29(5), 28(8).

EXAMPLE 15 Preparation of 7-Nitro-1,2,4-Benzotriazine 1,4-Dioxide viaReductive Deamination ##STR23##

To a stirred solution of t-butyl nitrite 988 mg, 0.85 mmol) in DMF (5ml) at 60° C. was added 7-nitro-3-amino-1,2,4-benzotriazine 1,4-dioxide(14) (38 mg, 0.17 mmol). After 30 min the addition of further t-butylnitrite (175 mg, 170 mmol) to the dark red slurry was immediatelyfollowed by a change in coloration and effervescence. After anadditional 10 min the orange solution was reduced to a red solid invacuo and chromatographed with 1% AcOH/CH₂ Cl₂ to give 3 mg of theproduct 27 as a yellow solid (10% yield). NMR δ(90 MHz, d₆ -dimethylsulfoxide) 7.68 (d, 1 H, J=9.2 Hz), 7.92 (dd, 1 H, J=9.2, 2.2 Hz), 8.10(d, 1 H, J=2.2 Hz), 8.65 (s, 1 H). UV: λ_(max) 420, 310, 240, 205. MS:m/z (relative intensity) 209(9), 208(100, M+), 192(54), 181(14),162(16), 105(9), 77(28), 75(52), 74(27), 63(21), 62(16), 30(77), 18(26).

EXAMPLE 16 3-ethyl-1,2,4-benzenetriazine-1,4-dioxide (31) ##STR24##

The hydrazone (28), formed from the condensation of propionaldehyde andphenyl hydrazine, was reacted with benzenediazonium chloride in amixture of acetic acid, sodium nitrite and HCl to give the formazan(29). Cyclization with BF₃ --AcOH (boron trifluoride-acetic acid) at90-95° C. gave 3-ethyl-1,2,4-benzenetriazine (30) as an oil, which waspurified by distillation. Oxidation with 70% hydrogen peroxide andtrifluoroacetic acid anhydride (TFAA) in CH₂ Cl₂ gave3-ethyl-1,2,4-benzenetriazine-1,4-dioxide (31). The title compound 31was purified using normal-phase column chromatography andrecrystallization from aqueous ethanol to give material of 99.8% purity.The melting point of 31 was found to be 141-142° C.

EXAMPLE 17 3-propyl-1,2,4-benzotriazine-1,4-dioxide (32)

3-propyl-1,2,4-benzotriazine-1,4-dioxide (32) was prepared and purifiedaccording to the method of Example 16 (preparation of3-ethyl-1,2,4-benzenetriazine-1,4-dioxide) except that the hydrazoneformed from the condensation of butyraldehyde and phenyl hydrazine wasused in the reaction with benzenediazonium instead of the hydrazoneformed from the condensation of propionaldehyde and phenyl hydrazine.The melting point of 32 was found to be 114-116° C.

EXAMPLE 18 3-(1-hydroxyethyl)-1,2,4-benzotriazine 1-oxide ##STR25##

3-chloro-1,2,4-benzotriazine 1-oxide was treated with a slight excess oftri-N-butylvinyltin in a mixture of acetonitrile, tritoluyl phosgene andtriethylamine, using palladium II catalysis (Pd II acetate) in a sealedtube at 100° C. for 48 hours. Removal of solvent and purification bycolumn chromatography gave 3-vinyl-1,2,4-benzotriazine 1-oxide (33).Reduction with 9-borabicyclo [3.3.1] nonane (9-BBN) followed byoxidation with sodium hydroxide and hydrogen peroxide gave the titlecompound 3-(1-hydroxyethyl)-1,2,4-benzotriazine 1-oxide (34).

EXAMPLE 19

Tirapazamine and cisplatin were tested in an in vivo RIF-1 tumor model.Tirapazamine and cisplatin were also tested in an in vitro assay usingRIF-1 cells under hypoxic and aerobic conditions.

Animals and tumors: The RIF-1 fibrosarcoma (developed and maintained inthe laboratory of Dr. Martin Brown, Department of Radiation Oncology,Stanford University, Stanford, Calif.; Twentyman et al. J. Nat'l CancerInst. 64: 595-604) in C3H/Km mice (bred and maintained by the RadiationBiology Division at Stanford University Medical School) housed underdefined flora conditions, was maintained alternately in vivo and invitro, according to a previously published protocol (Twentyman, supra).Tumor cell monolayers, growing in Waymouth's medium supplemented with15% fetal calf serum, were harvested with 0.05% trypsin. From thissuspension, 2×10⁵ cells in 0.05 ml medium were inoculated intradermallyin the back of each mouse at a site approximately 2 cm above the tail.Experiments were begun two weeks later when the mean tumor volume wasapproximately 200 mm³.

Drugs: Tirapazamine (SR 4233) was supplied by Sterling Drug Inc (NewYork, N.Y.). For animal studies, the drug was dissolved in normal salineat a concentration of 1 mg/ml and injected intraperitoneally (i.p.) on ammol/kg basis. Cisplatin (c-DDP) from Bristol Laboratories (Princeton,N.J.) was dissolved in sterile water and injected i.p. in 0.01 ml/gmbody weight.

Cell Survival: For animal studies, RIF-1 cell survival was evaluatedaccording to an in vivo/in vitro excision assay. Toward this end, micewere killed 24 hours after cisplatin treatment; tumors were excised,minced, and dissociated with an enzyme cocktail (Twentyman supra) andcells were plated for clonogenic assay. Resultant tumor cell colonieswere stained with crystal violet and counted after two weeks incubationat 37C in a 5% CO₂ humidifed atmosphere. Relative clonogenic cells pertumor was calculated as the product of plating efficiency and tumor cellyield for treated tumors relative to that for control untreated tumorsassayed in parallel.

For the studies on cells in vitro, RIF-1 cells were seeded into 60 mmglass petri dishes in Waymouth's medium supplemented with 15% fetalbovine serum at a concentration of 2×10⁴ cells per dish. The experimentswere performed 4 to 5 days later when there were approximately 10⁶ cellper dish at the time of treatment. The growth medium was then replacedwith 2 ml of medium without serum containing tirapazamine at aconcentration of either 2 or 4 μg/ml. In each experiment, groups wereincluded in which treatment with tirapazamine and cisplatin wereperformed both simultaneously and with an interval between the twotreatments. In those groups in which there was an interval immediatelyafter the exposure to tirapazamine, the cells were rinsed twice and themedium replaced with full growth medium until the time for the secondtreatment (with cisplatin), which was also performed in medium withoutserum. Both the exposure to tirapazamine and to cisplatin were for onehour under hypoxic conditions. To achieve hypoxia, the dishes wereloaded into specially fabricated, prewarmed aluminum gassing chamberswhich were placed in a shaking table and connected to a gassing manifoldcomprising a vacuum outlet line and inlet lines for air or nitrogen (+5%CO²). Hypoxia was achieved in the aluminum chambers through a series of5 alternate evacuations in 2 to 3 minutes to 0.1 atmosphere followed bygassing with nitrogen (+5% CO²). After gassing, the chambers were sealedand incubated for one hour at 37C. Measurement of the oxygen level inthe medium using a Clarke electrode showed that hypoxia was achievedrapidly (in approximately 10 minutes with an average pO₂ level duringthe one hour exposure of less than 200 parts/million oxygen).Immediately after the treatment with cisplatin, the cells weretrypsinized, counted and replated in plastic petri dishes in Waymouth'smedium supplemented with 15% fetal bovine serum and incubated for 14days at 37C in a 5% CO2 humidified atmosphere, after which the colonieswere stained with crystal violet and counted.

Normal Tissue: The response of normal tissue to tirapazamine andcisplatin was evaluated in the kidney and bone marrow through blood ureanitrogen (BUN) assays and peripheral white cell counts. Blood sampleswere taken from tail veins or by cardiac puncture. No anticoagulantswere used. Peripheral white cell counts for individual mice weredetermined from 20 μl whole blood diluted in 0.280 ml 3% acetic acid.For serum BUN assays, blood from two mice was pooled, coagulated,vortexed, and centrifuged at 830 g for 15 minutes. After the serum wasaspirated, BUN values were determined by a commercial clinicalveterinary laboratory. Survival to 30 days was also recorded in anotherexperiment.

Results:

(a) Results on tumors in vivo: FIG. 1 shows the pooled results from twoexperiments in which 0.35 mmol/kg tirapazamine (63 mg/kg) was deliveredto the tumor-bearing mice at various times over an interval from 3 hoursprior to 2 hours after delivery of 8 mg/kg cisplatin, and clonogenicsurvival was assessed at 24 hours. The x-axes shows the relative numberof clonogenic cells per tumor, The y-axis shows the time ofadministration of tirapazamine relative to administration of cisplatin(-2 hours represents data obtained from mice injected with tirapazaminetwo hours before the cisplatin injection). Open circles representtirapazamine alone; open squares represent cisplatin alone; closedsquares represent tirapazamine and cisplatin. As shown in FIG. 1, whentirapazamine is administered at intervals between three hours prior toor one to two hours after cisplatin, the relative numbers of clonogeniccells per tumor decreases from about 10-4 to about 10-7. These figuresrepresent a 10-fold to a 1,000-fold decrease in the number of clonogeniccells per tumor in comparison with the number of clonogenic cells pertumor when tirapazamine is administered at the same time as cisplatin.The synergistic effect of tirapazamine and cisplatin was most pronouncedwhen tirapazamine is administered from about three hours to one hourprior to cisplatin, with the greatest interactive effect being seen attwo and one half hours prior to administration of cisplatin.

Because of the large amount of cell killing observed at the nadir inFIG. 1 (which was on the border of the limits of the clonogenic assay),the dose of tirapazamine in the experiment was reduced from 0.35 mmol to0.27 mmol/kg (48.6 mg/kg) and the experiments were repeated. In thisexperiment, the time gap between administration of cisplatin andtirapazamine was extended to twenty-four hours. The results of thisexperiment are shown in FIG. 2. The x- and y-axes are the same as forFIG. 1. Open circles represent tirapazamine alone; open squaresrepresent cisplatin alone; closed squares represent cisplatin andtirapazamine. As shown in FIG. 2, the enhanced interaction betweentirapazamine and cisplatin was present when tirapazamine wasadministered up to twenty-four hours prior to cisplatin.

Despite the reduction in amount of tirapazamine administered in thesecond set of experiments, the data from the three experiments show thesame results: essentially additive toxicity when the drugs are giventogether, and a major cytotoxic interaction when the drugs are separatedin time with the maximal reduction in the number of clonogenic cells pertumor when tirapazamine treatment preceded cisplatin by approximately2.5 hours.

Additional experiments were performed with various doses of tirapazaminegiven 2.5 hours before either 4 or 8 mg/kg cisplatin. There was anapproximately exponential reduction in tumor cell survival at both dosesof cisplatin with increasing dose of tirapazamine.

(b) Results on Normal Tissue: Preliminary studies using C3H miceindicated that white cell counts reached a nadir on the third day aftertreatment with tirapazamine and cisplatin and then rose again to nearcontrol levels on day five. A dose response study was therefore done onday 3 of cisplatin alone and tirapazamine plus cisplatin with thetirapazamine dose (0.35 mmol/kg) given 2.5 hours before cisplatin.Cisplatin was administered at three different dose levels--10, 14, and18 mg/kg. Both tirapazamine and cisplatin produced a mild leukopenia andthe combination produced an affect equal to that predicted from addingthe responses to the individual drugs.

Assays of serum BUN were performed on the sixth day after injection oftirapazamine and cisplatin, based on a preliminary investigation of thetime for maximum increase in BUN following high doses of cisplatin. BUNin C3H mice six days after a single injection of tirapazamine (0.27mmol/kg), cisplatin (10, 14, or 18 mg/kg) or the two drugs giventogether (with tirapazamine injected two and one half hours before thecisplatin injection). BUN levels for doses of 10 and 14 mg/kg cisplatinalone were similar to BUN levels for untreated mice (approximately 30mg/dl). However, 18 mg/kg cisplatin alone had a BUN level of about 80mg/dl. By contrast, at each dose of the combination of drugs, the BUNlevel of the treated mice was less than the BUN levels of untreatedmice. These results show that tirapazamine in combination with cisplatindoes not add to cisplatin kidney toxicity and may even protect at thehighest dose tested.

As a further test of whether tirapazamine enhanced the systemic toxicityof cisplatin, an LD50 experiment was performed with cisplatin alone andcisplatin preceded 2.5 hours before injection by tirapazamine. The LD50for mice treated with 0.35 mmol/kg tirapazamine plus cisplatin was 17.7mg/kg (95% confidence limit: 16.8-18.7 mg/kg), as contrasted with thatfor cisplatin alone which was 17.8 (17.1-18.5 mg/kg).

(c) Results of in vitro experiments

Cells were exposed to a one hour period to tirapazamine (2 or 4 μg/ml)under hypoxic conditions and also exposed to cisplatin (2 μg/ml) for onehour as a function of time later. The concentration of each agent waschosen to produce a similar level of cell killing of hypoxic cells asthat with the RIF-1 tumors in vivo: for tirapazamine (0.3 and 0.009 andat 2 and 4 μg/ml, respectively) and for cisplatin (3.5×10⁻³). Eachexperiment contained groups in which there was no separation between theexposure of the two agents (i.e., tirapazamine and cisplatin wereadministered simultaneously for one hour under hypoxic conditions), aswell as a group in which the two exposures were separated from one tofour hours. The results obtained for the drugs given simultaneously werenot significantly different from the product of the survivals of the twoagents given separately (i.e., compatible with additivity); whereas whenthe drugs were separated, there was more cell killing by a factor of upto 10². There is a similar kinetics of enhancement of cell killing asobserved in the in vivo results, though the absolute magnitude of theeffect of splitting the two doses is less than that observed in vivo Tocheck that the interaction between the two agents depended on thepresence of hypoxia, the experiments were repeated with three hoursbetween exposure of the cells to tirapazamine under aerobic conditionsand exposure of the cells to cisplatin under hypoxic conditions. Inthese experiments, there was no cytotoxicity due to the tirapazamine,and there was no potentiation of the cell killing compared to thatproduced by cisplatin alone in the same experiments.

I claim:
 1. A method of treating a mammal having a solid tumor, saidmammal in need of such treatment, comprising(a) administering to saidmammal a synergistically effective amount of a compound having theformula ##STR26## n is 0 or 1; and Y¹ and Y² are independently either H;nitro; halogen; hydrocarbyl (1-14C) including cyclic and unsaturatedhydrocarbyl, optionally substituted with 1 or 2 substituents selectedfrom the group consisting of halogen, hydroxy, epoxy, alkoxy (1-4C),alkylthio (1-4C), primary amino (NH₂), alkyl (1-4C) secondary amino,dialkyl (1-4C) tertiary amino, dialkyl (1-4C) tertiary amino where thetwo alkyls are linked together to produce a morpholino, pyrrolidino orpiperidino, acyloxy (1-4C), acylamido(1-4C) and thio analogs thereof,acetylaminoalkyl (1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl,alkylcarbamyl (1-4C), alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C),wherein the hydrocarbyl can optionally be interrupted by a single ether(--O--) linkage; or wherein Y¹ and Y² are independently eithermorpholino, pyrrolidino, piperidino, NH₂, NHR', NR'R'O(CO)R', NH(CO)R',O(SO)R', or O(POR')R' in which R' is a hydrocarbyl (1-4C) which may besubstituted with OH, NH₂, alkyl (1-4C) secondary amino, dialkyl (1-4C)tertiary amino, morpholino, pyrrolidino, piperidino, alkoxy (1-4C), orhalogen substituents, or pharmacologically acceptable salt of saidcompound;and (b) administering to said mammal, from about one half hourto about twenty-four hours after administering said compound, aneffective amount of taxol.
 2. The method of claim 1 wherein said taxolis administered from about one hour to about eighteen hours afteradministering said compound.
 3. The method of claim 2 wherein said taxolis administered from about two to about three hours after administeringsaid compound.
 4. The method of claim 1 wherein said compound is3-amino-1,2,4-benzotriazine 1,4-dioxide.
 5. The method of claim 4wherein said 3-amino-1,2,4-benzotriazine 1,4-dioxide is administered tosaid mammal in amounts of from about thirty milligrams to about seventymilligrams per kilogram body weight of said mammal.
 6. A method ofincreasing the cytotoxicity of taxol towards a solid tumor, said tumorsusceptible to treatment with said taxol, comprising administering to amammal having such a tumor, from about one hour to about two hours afteradministering said taxol or from about one half hour to abouttwenty-four hours prior to administering said taxol, a synergisticallyeffective amount of a compound having the formula ##STR27## n is 0 or 1;and Y¹ and Y² are independently either H; nitro; halogen; hydrocarbyl(1-14C) including cyclic and unsaturated hydrocarbyl, optionallysubstituted with 1 or 2 substituents selected from the group consistingof halogen, hydroxy, epoxy, alkoxy (1-4C), alkylthio (1-4C), primaryamino (NH₂), alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiaryamino, dialkyl (1-4C) tertiary amino where the two alkyls are linkedtogether to produce a morpholino, pyrrolidino or piperidino, acyloxy(1-4C), acylamido(1-4C) and thio analogs thereof, acetylaminoalkyl(1-4C), carboxy, alkoxycarbonyl (1-4C), carbamyl, alkylcarbamyl (1-4C),alkylsulfonyl (1-4C) or alkylphosphonyl (1-4C), wherein the hydrocarbylcan optionally be interrupted by a single ether (--O--) linkage; orwherein Y¹ and Y² are independently either morpholino, pyrrolidino,piperidino, NH₂, NHR', NR'R'O(CO)R', NH(CO)R', O(SO)R', or O(POR')R' inwhich R' is a hydrocarbyl (1-4C) which may be substituted with OH, NH₂,alkyl (1-4C) secondary amino, dialkyl (1-4C) tertiary amino, morpholino,pyrrolidino, piperidino, alkoxy (1-4C), or halogen substituents, orpharmacologically acceptable salt of said compound.
 7. The method ofclaim 6 wherein said compound is 3-amino-1,2,4-benzotriazine1,4-dioxide.
 8. The method of claim 6 wherein said compound isadministered from about one hour to about eighteen hours prior toadministration of said taxol.
 9. The method of claim 8 wherein saidcompound is administered from about 2 hours to about 5 and one halfhours prior to administration of said taxol.